In vivo time-lapse fluorescence imaging of individual retinal ganglion cells in mice

Abstract

We have developed a technique that permits time-lapse imaging of retinal ganglion cells (RGCs), their dendritic arbors and their axons in mammals in vivo. This technique utilizes a standard confocal laser scanning microscope, transgenic mice that express yellow fluorescent protein (YFP) in a subset of RGCs and survival anesthesia techniques. The same individual RGCs with their dendritic arbors and axons were multiply imaged in vivo in both adult and juvenile mice. Additionally, the same RGC that was imaged in vivo could then be located and imaged in fixed retinal whole mount preparations. This novel technique has many potential applications.

Figure 1. Custom designed microscope stage with mouse

Top Left: top view of the aluminum stage with a central depressed 6 mm window for imaging. Top Middle: underside view with heating element. Top Right: mouse in imaging position. Lower Left: side view of aluminum stage. Lower Middle: oblique view of aluminum stage. Lower Right: mouse in imaging position; note glass coverslip.

Figure 2. Subset of retinal ganglion cells (RGCs) expresses yellow fluorescent protein (YFP)

This is a montage of a whole mount fixed adult mouse retina from the YFP-H transgenic line. Typically 50 – 100 RGCs are YFP positive. The intensity of YFP fluorescence can vary among the positive neurons. Acquisition settings: 5x objective, 0.7 zoom, stack size: 2606 μm x 2606 μm x 127–211 μm, pixel size: 2.54 μm x 2.54 μm x 21.1 μm, laser power 10%, pinhole 67 μm, pixel time 3.2 μs, projection, scale bar 200 μm.

Figure 3. YFP-positive retinal ganglion cell

This is a single YFP-positive RGC in an adult fixed whole mount retina. The dendritic arbor and boutons are appreciated as well as the axon exiting the field of view superiorly. Left: en face view. Right: 90 degree view. Acquisition settings: 20x objective, no zoom, stack size: 461 x 461 x 26.5 μm, pixel size: 0.45 x 0.45 x 0.95 μm, pixel time 1.6 μs, laser power 10%, pinhole 61 μm, projections, scale bars: 100 μm (left) and 20 μm (right).

Figure 4. Multiple RGC morphologies and rare amacrine cell in fixed whole mount

Various morphologic subtypes of RGCs are YFP-positive in these mice. Of approximately 500 YFP-positive retinal neurons screened, 2 non-RGCs were found, one of which is located inferiorly in this image. It appears to be a bistratified amacrine cell (See Figure 5). Left: en face view. Right: 90 degree view. Acquisition settings: 20x objective, no zoom, stack size: 461 x 461 x 38 μm, pixel size: 0.45 x 0.45 x 1 μm, pixel time 1.6 μs, laser power 10%, pinhole 61 μm, projections, scale bars: 50 μm (left) and 20 μm (right).

Figure 5. Rare YFP-positive amacrine cell

This is the same amacrine cell imaged in Figure 2 in whole mount fixed retina. Its dendritic tree is bistratified and is one of only 2 found out of approximately 500 screened YFP-positive neurons. The second amacrine cell had a similar morphology (data not shown). Left: en face view. Right: 90 degree view. Acquisition settings: 40x oil objective, 3x zoom, stack size: 77 x 77 x 33 μm, pixel size: 0.07 x 0.07 x 1 μm, pixel time 1.6 μs, laser power 10%, pinhole 71 μm, projections, scale bars: 20 μm (left) and 20 μm (right).

Figure 6. In vivo imaging of RGC axons emanating from optic disc in adult mouse

These are images taken in vivo from the right eye of an adult mouse showing multiple YFP-positive axons entering the optic nerve head and rare RGC bodies in the peripapillary region. Acquisition settings: 5x objective, no zoom, single image planes, pixel time 1.6 μs, laser power 53%, pinhole 1000 μm.

Figure 7. In vivo imaging of RGC dendritic arbor

Using a 10x Plan-Neo/0.3 NA objective, this RGC’s dendritic arbor details were imaged in vivo (See also Figure 6). Acquisition settings: 10x objective, no zoom, single image plane, pixel time 1.6 μs, laser power 51%, pinhole 545 μm.

Figure 8. Single RGC imaged in vivo then in fixed whole mount

Soon after anesthesia, this animal stopped breathing. Therefore these in vivo images, taken immediately after cessation of breathing, do not have breathing movement artifacts. Z stacks of images were thereby obtained. The eye was then fixed, and the same RGC was imaged in whole mount. Left panel acquisition settings: 5x objective, no zoom, z stack, projection, pixel time 1.6 μs, laser power 10%, pinhole 294 μm. Right panel: 20x objective, no zoom, stack size: 461 x 461 x 28 μm, pixel size: 0.45 x 0.45 x 1 μm, pixel time 1.6 μs, laser power 10%, pinhole 61 μm, projection, scale bar: 50 μm; fixed image inverted to match orientation of in vivo image.

Figure 9. In vivo imaging in juvenile mouse

In this 4 week old mouse image, 4 RGC bodies can be appreciated. Dendritic details are apparent in 2 of the RGCs. Acquisition settings: 5x objective, no zoom, single image plane, pixel time 1.6 μs, laser power 52%, pinhole 761 μm.

Figure 10. In vivo time-lapse imaging in juvenile mouse

This 4 week juvenile mouse was imaged at day 0 (left), and then again on day 2 (right). 3 RGCs are appreciated in this peripapillary region. Acquisition settings: 5x objective, no zoom, single image plane, pixel time 1.6 μs; Day 0: laser power 51%, pinhole 454 μm; Day 2: laser power 52%, pinhole 670 μm.

Figure 11. In vivo time-lapse imaging in adult mouse

This region of the retina in an adult mouse was imaged in vivo, the animal was survived then imaged again 2 days later. The 2 central RGCs have differing dendritic morphologies which are grossly stable over the 2 day interval. Acquisition settings: 5x objective, no zoom, single image plane, pixel time 1.6 μs; Day 0: laser power 53%, pinhole 1000 μm; Day 2: laser power 51%, pinhole 636 μm.

Figure 12. In vivo time-lapse imaging of single RGC then imaged in whole mount

This RGC was imaged initially in vivo, the animal was survived and then imaged again the next day in vivo, after which the same RGC was imaged in fixed whole mount preparation the same day. There appears to be some artifactual stretching of the tissue in the fixed preparation, as the distance between the dendritic arbor and the nearby axons is increased relative to the in vivo image. By utilizing low power views of the entire retina as a map we are assured that the fixed cell is the same as that imaged in vivo. Left panel acquisition settings: 5x objective, no zoom, single image plane, pixel time 1.6 μs, laser power 51%, pinhole 636 μm. Center panel: 5x objective, no zoom, single image plane, pixel time 1.6 μs, laser power 52%, pinhole 761 μm. Right panel: 20x objective, no zoom, stack size: 461 x 461 x 34 μm, pixel size: 0.45 x 0.45 x 3.8 μm, pixel time 1.6 μs, laser power 15%, pinhole 61 μm, projection, scale bar: 100 μm; fixed image inverted & rotated to match orientation of in vivo images.